The recent advances in the field of biotechnology have required faster and more accurate techniques for recovery, purification and analysis of biological and biochemical substances, such as proteins. Electrophoresis and chromatography are two commonly used such techniques.
In electrophoresis, charged particles are separated by migration in an electric field. More specifically, a sample is placed on a soft solid support medium, such as an agarose or polyacrylamide gel slab, which in turn is placed between two electrodes, a positively charged anode and a negatively charged cathode. As the current is switched on, each component of the sample will migrate at a characteristic rate determined by its net charge and its molecular weight. One essential property of a well working electrophoresis gel is its melting point, which affects the ability to extract the migrated target compounds from separate gel spots. Thus, a low melting point gel is commonly advantageous. Native agarose is commonly used in electrophoresis gels, but they have been noted to involve some problems. For example, even though the coarse pore structure of the native agarose is excellent for resolving large macromolecules, for smaller molecules smaller molecular weight agarose must be prepared. This is commonly obtained by increasing the agarose content of the gel, which however produces high viscosities in the solutions rendering casting of gels thereof difficult. To overcome such problems and others, modified agarose has been suggested for electrophoresis gels.
Electrophoresis is discussed in U.S. Pat. No. 3,956,273 (Guiseley), which relates to agarose or agar compounds useful for electrophoresis or diffusive interactions, but also as thickeners. The compounds have been modified with alkyl and alkenyl groups in order to lower their gelling and melting temperatures, and to increase their clarity as compared to the unmodified material. More specifically, the agar or agarose is first dissolved in strong alkali, after which a suitable reagent is added to provide the modification. A difunctional agent such as epichlorohydrin may be used, but only under conditions which prevent cross-linking.
Electrophoresis is also discussed in U.S. Pat. No. 5,143,646 (Nochumson et al), which relates to electrophoretic resolving gel compositions comprising polysaccharide hydrogels, such as agarose, which has been derivatised and depolymerised sufficiently to reduce its casting-effective viscosity. The disclosed compositions do not require any cross-linking or polymerising agents.
Further, U.S. Pat. No. 5,541,255 (Kozulic) relates to gels for electrophoresis, and more specifically to cross-linked linear polysaccharide polymers. The gels are formed by dissolving a polysaccharide in a solvent such as water; adding a cross-linking agent, which is not charged nor which becomes charged upon contact with water; and incubating the mixture in a quiescent state to simultaneously react the polysaccharide and the cross-linking agent and to gel the product into a slab. According to U.S. Pat. No. 5,541,255, the prior art electrophoresis gels could be redissolved by water, while the U.S. Pat. No. 5,541,255 invention provides a gel which is water insoluble. These properties are obtained due to the simultaneous cross-linking and gelation, and also due to the high ratio of cross-linker to polysaccharide.
In chromatography, two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample. In liquid chromatography, a liquid sample, optionally combined with a suitable buffer constitutes the mobile phase, which is contacted with a stationary phase, known as a separation matrix. Usually, the matrix comprises a support to which ligands, which are groups capable of interaction with the target, have been coupled.
Separation matrices are commonly based on supports made from inorganic materials, such as silica, or organic materials, such as synthetic or natural polymers, or the like. The synthetic polymers, such as styrene and divinylbenzene, are often used for supports that exhibit some hydrophobicity, such as size exclusion chromatography, hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC). Further, the synthetic polymers are sometimes preferred over natural polymers due to their flow properties, which may be more advantageous since synthetic polymers are often more rigid and pressure-resistant than the commonly used natural polymer supports.
The natural polymers, which are commonly polysaccharides such as agarose, have been utilised as supports of separation matrices for decades. Due to the presence of hydroxyl groups, the surfaces of the natural polymers are usually hydrophilic, giving essentially no non-specific interactions with proteins. Another advantage of the natural polymers, which is of specific importance in the purification of drugs or diagnostic molecules for internal human use, is their non-toxic properties. Agarose can be dissolved in water at increased temperature, and will then form a porous gel upon cooling to a certain temperature (the gelling point). On heating, the gel will melt again at a temperature (the melting point), which is usually considerably higher than the gelation point. The gelation involves helix-helix aggregation of the polysaccharide polymers, and is sometimes referred to as a physical cross-linking. To optimise the target mass transport rate and the area with which the target interacts, it is often desired to increase the porosity of the support, which can be achieved by varying the agarose concentration. However, another essential parameter to consider is the flow properties of the support. The matrix is normally used in the form of a packed bed of particles (spherical or non-spherical). When the mobile phase is forced through the bed, the back pressure of the bed will mainly be controlled by the interstitial channels between the particles. At low flow rates, the particles can be regarded as incompressible and then the back pressure increases linearly with the flow rate, with the slope depending on the particle size. At higher flow rates, the particles may start to deform under the hydrostatic pressure, resulting in diminishing diameters of the interstitial channels and a rapidly increasing back pressure. At a certain flow rate, depending on the rigidity of the matrix, the bed will collapse and the back pressure approaches infinity unless it is switched off automatically by the chromatography system. To improve the rigidity and hence the flow properties of agarose, it is frequently cross-linked. Such cross-linking takes place between available hydroxyl groups, and may be obtained e.g. with epichlorohydrin.
U.S. Pat. No. 4,973,683 (Lindgren) relates to the cross-linking of porous polysaccharide gels, and more specifically to a method of improving the rigidity while minimising the non-specific interaction of a porous polysaccharide gel. The method involves providing an agarose gel and a reagent denoted “monofunctional”, which comprises a reactive group, such as a halogen group or an epoxide group, and a double bond. The reagent is bound to the gel via its reactive group; and the double bond is then activated into an epoxide or halohydrin, which is finally reacted with hydroxyl groups on the agarose to provide cross-linking.
U.S. Pat. No. 5,135,650 (Hjertén et al) relates to highly compressible chromatographic stationary phase particles, such as agarose beads, which are sufficiently rigid for HPLC and non-porous to the extent that it is impenetrable by solutes. More specifically, such beads are produced by starting from porous agarose beads, which are contacted with an organic solvent to collapse the porosity, after which the bead surfaces inside the collapsed pores are cross-linked to fix the pores in their collapsed state. Alternatively, the beads are produced by filling the pores with a polymerisable substance, which grafts to the pore surfaces, and performing graft polymerisation. One stated advantage of the invention disclosed is that a single stationary phase is effective at high pressures and yet can be used at low pressures.
U.S. Pat. No. 6,602,990 (Berg) relates to a process for the production of a porous cross-linked polysaccharide gel, wherein a bifunctional cross-linking agent is added to a solution of polysaccharide and allowed to bind via its active site to the hydroxyl groups of the polysaccharide. A polysaccharide gel is then formed from the solution, after which the inactive site of the cross-linking agent is activated and cross-linking of the gel performed. Thus, the cross-linking agent is introduced into the polysaccharide solution, contrary to the above discussed methods wherein it is added to a polysaccharide gel. The bifunctional cross-linking agent comprises one active site, i.e. a site capable of reaction with hydroxyl groups of the polysaccharide, such as halides and epoxides, and one inactive site, i.e. a group which does not react under the conditions where the active site reacts, such as allyl groups. Thus, the present bifunctional cross-linking agent corresponds to the “monofunctional reagents” used according to the above-discussed U.S. Pat. No. 4,973,683 (Lindgren). Particles comprised of the resulting gel have been shown to present an improved capability of withstanding high flow rates and back pressures. A drawback with the U.S. Pat. No. 6,602,990 method is that bromine is required for the activation of the cross-linking agent.
Finally, U.S. Pat. No. 5,998,606 (Grandics) relates to a method of synthesising chromatography media, wherein cross-linking and functionalisation of a matrix takes place simultaneously. More specifically, double bonds provided at the surface of a polymeric carbohydrate matrix are activated in the presence of a metallic catalyst to cross-link the matrix and functionalise it with halohydrin, carboxyl or sulphonate groups. The double bonds are provided at the matrix surface by contact with an activating reagent, which contains a halogen atom or epoxide and a double bond. Thus, the U.S. Pat. No. 5,998,606 activating reagent corresponds to the U.S. Pat. No. 4,973,683 monofunctional reagent and the U.S. Pat. No. 6,602,990 bifunctional cross-linking agent.
Thus, even though there are a number of techniques available for producing cross-linked polysaccharide separation matrices, since different applications will put different requirements on the matrix, there is still a need within this field of alternative methods.